Syeling Lai

Expression of p16, Rb, and cyclin D1 gene products in oral and laryngeal squamous carcinoma: biological and clinical implications.

Cyclin D1, p16, and Rb genes play a critical role in the regulation of the G1-S transition of the cell cycle and are frequently altered in several neoplastic entities. Analysis of the protein products of these genes by molecular and immunohistochemical methods provides information on their functional status and allows for the phenotypic evaluation of tumor cells. We performed Western blotting and immunohistochemical analysis on tissues from 35 primary oral and laryngeal squamous carcinoma specimens with previous molecular analysis of the p16 gene and correlated the results with relevant clinicopathologic factors. Our study shows significant concordance between Western blotting and immunostaining results for cyclin D1 (P = .01), p16 proteins (P = .01), and Rb (P = .04). Heterogeneous staining of tumor cells and the positivity of non-neoplastic host elements for Rb by immunohistochemistry contributed to the discrepancy noted in some tumors by Western blotting. Significant reciprocal relationship between p16 and Rb proteins was observed (P < .001); in most tumors, absence of p16 (89%) and detectable Rb (94%) proteins were found. Two tumors had negative cyclin D1 expression, and one third overexpressed this protein. There was a lack of correlation between cyclin D1 overexpression and the clinicopathologic factors studied. Our results indicate that the absence of p16 in most of these tumors may constitute an early tumorigenic event and that the loss of the Rb function plays a minor role in HNSC.

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA

IntroductionSPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.MethodsIn in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Massons trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays.ResultsSPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-β receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues.ConclusionsSpecific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

Differential expression profiling of head and neck squamous carcinoma: Significance in their phenotypic and biological classification

The genetic events associated with the development and progression of head and neck squamous carcinoma (HNSC) are largely unknown. We analysed 12 matched pairs of histologically normal squamous mucosa and tumor specimens from six conventional and six phenotypic variants HNSC to define the differentially expressed genes in these tumors. Parallel expression analysis of 8055 unique genes was performed, and the level of the hybridization signal for each gene was measured after normalization. Hierarchical cluster analysis of the expressed genes showed distinct inter- and intra-tumoral patterns in and between conventional squamous carcinoma and squamous carcinoma variants. We also identified 26 (0.32%) differentially expressed genes that were consistently different between matched pairs of normal and tumor specimens; a selected set of the overexpressed genes was validated using real-time quantitative RT–PCR. The majority of the genes were associated with differentiation and proliferation. Our study defines a set of genes that could form the basis for the construction of limited HNSC targeted expression array and indepth studies and further highlights gene profile differences that may be useful in pathobiologic classification of HNSC.

Frequent loss of imprinting at the IGF2 and H19 genes in head and neck squamous carcinoma

Genomic imprinting is an inherited epigenetic phenomenon that results in parental – origin – specific gene expression in somatic cells. Relaxation or loss of this feature in certain genes has been demonstrated in several pediatric and adult neoplasms, suggesting an association with tumorigenesis. We analysed 64 primary untreated head and neck squamous carcinoma for the loss of imprinting in the IGF2 and H19 genes to determine the implications of this alteration in the development and progression of these tumors. Forty-nine (77%) of the 64 tumors were informative for imprinting analyses of these genes. IGF2 and H19 were imprinted in all normal squamous epithelium examined. Twelve (37.5%) of 32 tumors informative for H19 and 11 (40.7%) of 27 tumors informative for IGF2 manifested loss of imprinting. Ten tumors were informative for both genes, of which four maintained the constitutional imprinting and six showed loss of imprinting at either H19 or IGF2. These data suggest that loss of imprinting at the IGF2 and H19 loci play a role in the oncogenesis of head and neck carcinoma.

Cyclooxygenase-2 protein reduces tamoxifen and N-(4-hydroxyphenyl)retinamide inhibitory effects in breast cancer cells.

Approximately 30–40% of estrogen receptor α (ERα)-positive breast tumors express high levels of the cyclooxygenase-2 (COX-2) protein, and these high levels have been associated with a poorer prognosis in breast cancer patients. We speculate that high levels of COX-2 induce drug resistance in ERα-positive breast tumors, thus reducing the survival rate of patients with such tumors. Human breast cancer cell lines that express high levels of COX-2 are generally ERα negative. To determine whether COX-2 induces drug resistance, plasmids encoding the COX-2 gene were stably transfected into ERα-positive MCF-7 human breast cancer cells (MCF-7/COX-2). MCF-7/COX-2 cells were resistant to the selective estrogen receptor modulator tamoxifen but not to its analog, raloxifene. MCF-7/COX-2 cells were also resistant to the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) but not to its analog, all-trans retinoic acid. In contrast, the sensitivities of MCF-7/COX-2 cells to doxorubicin and paclitaxel were similar to those of the parental MCF-7 cells. We then determined which COX-2 product, prostaglandin E2 (PGE2) or prostaglandin F2α is involved in the COX-2-mediated drug resistance. PGE2, but not PGF2α, blocked the antiproliferative effects of tamoxifen and 4-HPR. Agonists that activate PGE2 receptors and their downstream kinase effectors, protein kinases A and C, also blocked the growth inhibitory effects of these drugs. Increased levels of Bcl-2 and Bcl-XL proteins have been reported in mammary tumors of COX-2 transgenic mice and in human colon cancer cell lines that have high levels of COX-2. However, we did not observe any changes in Bcl-2, Bcl-XL, or Bax expression induced by COX-2 or PGE2. Here we report the novel findings that COX-2 uses PGE2 to stimulate the activities of protein kinases A and C to induce selectively tamoxifen and 4-HPR resistance in ERα-positive breast cancer cells.

Lack of Association of the CD247 SNP rs2056626 with Systemic Sclerosis in Han Chinese

Systemic sclerosis (SSc) is a complex disease involving multiple genetic factors. A recent genome-wide association study (GWAS) indicated that CD247 was strongly associated with SSc, which was subsequently confirmed in a SSc cohort of European population. However, genetic heterogeneity in different ethnic populations may significantly impact the complex trait of SSc. The studies herein aimed to examine whether the SSc-associated SNP rs2056626 of CD247 identified in Caucasian is also associated with Han Chinese SSc. A Han Chinese cohort consisting of 387 SSc patients and 523 healthy controls were examined in the studies. TaqMan assays were performed to examine the SNP. Exact p-values were obtained (Fisher’s test) from 2x2 tables of allele counts and disease status. The results showed that there was no association between rs2056626 of CD247 and SSc or any SSc subtypes of Han Chinese. The negative results are important in understanding genetics of SSc in different ethnic populations, which further suggest complex nature of genetics of SSc.

Association of the IRF5 SNP rs2004640 with systemic sclerosis in Han Chinese.

Systemic sclerosis (SSc) is a complex disease involving multiple genetic factors. An association of the IRF5 polymorphism with SSc was reported in Caucasian populations of Europe and North America, as well as in Japanese populations. The present study aimed to examine whether the SSc-associated SNP rs2004640 of IRF5 gene confer susceptibility to SSc and clinical features of SSc in a Han Chinese population. A Han Chinese cohort consisting of 424 SSc patients and 502 healthy controls were examined in the study. TaqMan assays were carried out to examine the SNP. Exact p-values were obtained (Fishers test) from 2×2 tables of allele counts and disease status. SSc patients of Han Chinese showed increased homozygous TT genotype of the rs2004640 (p = 0.027, odds ratio (OR) = 1.4, CI =1.03–1.93), which was significantly associated with pulmonary fibrosis of SSc and ATA-positive SSc of Han Chinese. The lcSSc and ACA-positive SSc of Han Chinese appeared also in association with the increased T allele frequency. However, the Chinese dcSSc did not show any association with the rs2004640. The results were consistent with previous reports in other ethnic populations in supporting the notion that polymorphisms of IRF5 may play an important role in susceptibility to SSc.

Hemophagocytic lymphohistiocytosis associated with influenza A (H1N1) infection in a patient with chronic lymphocytic leukemia: an autopsy case report and review of the literature.

H1N1 influenza A virus can trigger fatal hemophagocytic lymphohistiocytosis in immunocompromised patients and in immunocompetent hosts, usually children. We present a case of a 50-year-old man with low-burden chronic lymphocytic leukemia who had sudden reactivation of his leukemia triggered by influenza A (H1N1) infection with hemophagocytic lymphohistiocytosis during the 2009 H1N1 pandemic. His rapid course was complicated by acute respiratory distress syndrome with diffuse alveolar damage, a 6-fold rise in lymphocyte count, disseminated intravascular coagulation, and, ultimately, cardiac arrest. Major findings at autopsy included: bilateral H1N1 pneumonitis with diffuse alveolar damage, intra-alveolar pulmonary hemorrhage, pulmonary microthromboemboli, pulmonary hemorrhagic infarction, hemophagocytic lymphohistiocytosis in multiple locations, and diffuse chronic lymphocytic leukemia. Hemophagocytic lymphohistiocytosis is a serious and often fatal condition, which may be primary or secondary. It may be associated with high-grade lymphoproliferative malignancies, especially in patients with therapy-related leukocytopenia, but only rarely is it seen in uncomplicated chronic lymphocytic leukemia. Hemophagocytic lymphohistiocytosis may be triggered by a variety of infections (viral, fungal, bacterial and parasitic), but H1N1 influenza A-associated hemophagocytic lymphohistiocytosis is often rapidly fatal, especially in children. This adult patients clinical presentation with low tumor burden and leukocytosis is thus unique. We review the recently published autopsy findings in fatal influenza A (H1N1) infection and the association with resultant secondary hemophagocytic lymphohistiocytosis.

Loss of retinoblastoma gene function and heterozygosity at the RB locus in renal cortical neoplasms.

Alteration of the retinoblastoma (RB) gene, located on chromosome 13q14, has been implicated in the pathogenesis and biological behavior of several human cancers. We investigated the RB gene status by Western blotting and immunohistochemical analysis, as well as loss of heterozygosity (LOH) at the RB locus in 21 primary human renal neoplasms (including 3 oncocytomas). In only 1 of 21 tumors was there a discrepancy between Western blot and immunochemical staining. Overall, LOH was noted in 6 of 12 informative cases. However, only one of the tumors with LOH at the RB locus had loss of RB protein expression by both Western blot and immunohistochemical analysis. Loss of RB function was found in 4 of 18 carcinomas and in none of 3 oncocytomas as determined by absent RB nuclear staining in tumor cells. LOH at chromosome 13q14 was more noted in high-grade, DNA aneuploid, high-stage tumors and in patients with poor outcome. These results imply that (1) there is likely another tumor-suppressor gene on chromosome 13 involved in renal carcinogenesis, (2) LOH at chromosome 13q loci may be associated with aggressive behavior, and (3) the loss of RB function may have a role in a subset of renal carcinomas.

Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

IntroductionSumoylation is involved in nucleolus-nucleoplasm transport of DNA topoisomerase I (topo I), which may associate with changes of cellular and topo I functions. Skin fibroblasts of patients with systemic sclerosis (SSc) exhibit profibrotic cellular changes. The aims of this study were to examine the catalytic function and sumoylation of topo I in the nuclei of SSc fibroblasts, a major cell type involved in the fibrotic process.MethodsEleven pairs of fibroblast strains obtained from nonlesional skin biopsies of SSc patients and age/sex/ethnicity-matched normal controls were examined for catalytic function of nuclear topo I. Immunoprecipitation (IP)-Western blots were used to examine sumoylation of fibroblast topo I. Real-time quantitative RT-PCR was used to measure transcript levels of SUMO1 and COL1A2 in the fibroblasts.ResultsTopo I in nuclear extracts of SSc fibroblasts generally showed a significantly lower efficiency than that of normal fibroblasts in relaxing equivalent amounts of supercoiled DNA. Increased sumoylation of topo I was clearly observed in 7 of 11 SSc fibroblast strains. Inhibition of SUMO1 with SUMO1 siRNA improved the catalytic efficiency of topo I in the SSc fibroblasts. In contrast, sumoylation of recombinant topo I proteins reduced their catalytic function.ConclusionsThe catalytic function of topo I was decreased in SSc fibroblasts, to which increased sumoylation of topo I may contribute.